Amino acid nitrogen isotopic compositions show seep copepods gain nutrition from host animals

<jats:p>Deep-sea chemosynthetic ecosystems harbour high biomass centred around animals with chemoautotrophic symbionts. Despite being intensively studied over the last 4 decades, microscopic animals associated with and/or parasitic on dominating holobionts remain understudied. Here, we combine bulk tissue isotope analysis for carbon and nitrogen and compound-specific isotope analysis of amino acids (CSIA-AA) for nitrogen to elucidate the relationship between 2 copepod-host pairs from the Off Hatsushima hydrothermal seep in Sagami Bay, Japan: the vesicomyid clam <jats:italic>Phreagena okutanii</jats:italic> and <jats:italic>Hyphalion sagamiense</jats:italic> living on its mantle, and the tubeworm <jats:italic>Lamellibrachia columna</jats:italic> and <jats:italic>Dirivultus kaiko</jats:italic> found on its plume. Bulk tissue isotope analyses found overall large variations in δ<jats:sup>13</jats:sup>C and δ<jats:sup>15</jats:sup>N values across both associations, and did not allow conclusions on the trophic relationship between each pair. In contrast, CSIA-AA for <jats:italic>Phreagen</jats:italic><jats:italic>a-</jats:italic><jats:italic>Hyphalion</jats:italic> clearly showed trophic positions (TP<jats:sub>Glu/Phe</jats:sub>) increasing from gill (symbiont) to adductor muscle (host tissue) to <jats:italic>H. sagamiense</jats:italic>. In the case of <jats:italic>Lamellibrachi</jats:italic><jats:italic>a-</jats:italic><jats:italic>Dirivultus</jats:italic>, a similar increase in TP<jats:sub>Glu/Phe</jats:sub> was found from the plume to <jats:italic>D. kaiko</jats:italic>. These results show that both <jats:italic>H. sagamiense</jats:italic> and <jats:italic>D. kaiko</jats:italic> are nutritionally dependent on their respective hosts and therefore should be considered parasites despite being from copepod families that are typically not recognised as parasitic. Our CSIA-AA results represent the first use of this technique to document host-parasite relationships in chemosynthetic ecosystems. Understanding the role of parasites is of great importance in reconstructing energy flow in ecosystems, and our results underscore the promising nature of CSIA-AA in revealing their otherwise hidden relationships.</jats:p>